T cell inhibitory receptor compositions and uses thereof

ABSTRACT

The invention relates to compositions which bind T cell inhibitory receptor molecules and modulate T cell activity, and methods of using such compositions. Such compositions include biliary glycoprotein binding agents. Methods for modulating killer T cell activities, including cytotoxicity and proliferation also are provided.

RELATED APPLICATIONS

This application is a continuation of Ser. No. 11/049,270, filed11/049,270, now pending, which is a continuation of Ser. No. 09/884,196,filed Jun. 19, 2001, now issued as U.S. Pat. No. 6,852,320, which is adivisional of 09/293,504, filed Apr. 15, 1999, now abandoned, and claimspriority under 35 U.S.C. §119(e) from U.S. provisional patentapplication Ser. No. 60/081,895, filed Apr. 15, 1998, the entiredisclosures of which are incorporated herein by reference.

GOVERNMENT SUPPORT

This work was funded in part by the National Institutes of Health underGrant Numbers DK44319 and DK51362. The government may retain certainrights in this invention.

FIELD OF THE INVENTION

The invention relates to compositions which bind T cell inhibitoryreceptor molecules and modulate T cell activity.

BACKGROUND OF THE INVENTION

The biologic role of human intestinal intraepithelial lymphocytes (iIEL)and their functional relationship with the intestinal epithelial cell(IEC) remains incompletely characterized. Human iIELs have been variablyshown to exhibit cytolytic function and possibly immunoregulatoryfunction through the secretion of a variety of cytokines (Ebert, Clin.Exp. Immunol., 82:81-85, 1990; Lundqvist et al., J. Immunol.,157:1926-1934, 1996; Balk et al., Science, 253:1411-1415, 1991). Whetherthese functional activities are related to processes that may be uniqueto the gut associated lymphoid tissue such as oral tolerance and localimmunosurveillance against IEC injury and neoplastic transformation is,however, unclear. Moreover, the molecules on the cell surface of iIELsand their IEC counterreceptors which regulate the functional activationof iIELs and which may be utilized in this special microenvironment areonly beginning to be elucidated.

A significant fraction of human iIELs of both the small and largeintestine are CD8-αβ⁺ and CD45RO⁺ T cells which express a limited arrayof αβ and, to a lesser extent, γδ-T cell receptors (TCR) (Balk et al,1991; Jarry et al., Eur. J. Immunol., 20:1097-1103, 1990; Blumberg etal., J. Immunol., 150:5144-5153, 1993; Van Kerckhove et al., J. Exp.Med., 175:57-63, 1992; Chowers et al., J. Exp. Med., 180:183-190, 1994).These phenotypic properties indicate that iIELs are memory cells whichlocalize to the basolateral surface of IECs for the recognition of alimited number of antigens in the context of major histocompatibilitycomplex (MHC) class I or class I-like molecules on the IEC. However, themajority of iIELs in mouse and human are CD28⁻ suggesting that othercostimulatory molecules for TCR/CD3 complex-mediated activation may beimportant in providing necessary secondary signals for iIEL activation(Gelfanov et al., J. Immunol, 155:76-82, 1995; Gramzinski et al., Int.Immunol. 5:145-153, 1993; Russell et al., J. Immunol., 157:3366-3374,1996). Candidate costimulatory molecules for human iIELs include CD2(Ebert, Gastroenterology, 97:1372-1381, 1989), CD101 (Russell et al.,1996), BY-55 (Anumanthan et al., J. Immunol., 161:2780-2790, 1998) andα^(E)β₇ (Parker et al., Proc. Natl. Acad. Sci., 89:1924-1928, 1992)which are expressed by the majority of iIELs.

It has also become increasingly evident that in addition to activatingcostimulatory molecules, T cells can express a variety of molecules thatdeliver an inhibitory signal such that either the initial activation ofthe T cell is prevented or the activated state is downregulated. Theformer include the killer inhibitory receptors (KIR) which are expressedon a subset of T cells and bind specific types of majorhistocompatibility complex (MHC) class I molecules on the target cells(Lanier et al., Immunology, 7:75-82, 1995). The latter includes CTLA-4(CD 152) which, when expressed after T cell activation, binds eitherCD80 (B7.1) or CD86 (B7.2) on antigen presenting cells (Walunas et al.,J Exp. Med., 183:2541-2550, 1996; Krummel et al., J Exp. Med.183:2533-2540, 1996). These inhibitory receptors characteristicallycontain immunoglobulin-like domains extracellularly and one or moreimmune receptor tyrosine-based inhibitory motifs (ITIM) in theircytoplasmic tails which consists of the consensus sequence I/L/VxYxxL/V(SEQ ID NO:5) (Véy et al., J Immunol., 159:2075-2077, 1997). In the caseof CTLA-4, the ITIM is slightly modified to GxYxxM (SEQ ID NO:6)(Cambier, Proc. Natl. Acad. Sci., 94:5993-5995, 1997). ITIM-containingreceptors function in the recruitment of either the Src homologydomain-containing protein tyrosine phosphatases, SHP-1 and SHP-2, or theSH2 domain-containing inositol polyphosphate 5-phosphatase, SHIP(lsakov, Immunol. Res., 16:85-100, 1997). These phosphatases function inthe dephosphorylation of signaling molecules recruited by immunereceptor tyrosine-based activation motif (ITAM) bearing receptors likethose contained in the CD3-γ,δ,ε and ζ chains that associate with theTCR. As such, ITIM bearing receptors on T cells are predicted todownregulate activation events elicited by ITAM bearing receptors ifboth are ligated in close proximity to one another. Importantly, neitherCTLA-4 nor CD80/CD86 have been observed on human iIELs or IECs of thesmall intestine, respectively.

SUMMARY OF THE INVENTION

It has now been discovered that biliary glycoprotein (BGP; also known asCD66a and C-CAM), a member of the carcinoembryonic antigen family (CEA),is an inhibitory receptor for activated T cells contained within thehuman intestinal epithelium. These studies suggest that, in a regionalmicroenvironment that is predominantly CD28/CTLA4-CD80/CD86 negative,other receptor-ligand interactions may provide necessary downregulatorysignals to limit T cell activation and immunopathology.

According to one aspect of the invention, methods for enhancingspecifically the cytotoxicity or proliferation of killer T cells in asubject are provided. The methods include administering to a subject inneed of such treatment an agent that selectively reduces cross-linkingof biliary glycoprotein polypeptides in an amount effective to enhancethe cytotoxicity or proliferation of killer T cells in the subject. Incertain embodiments the agent is an antibody or antibody fragment whichbinds only a single biliary glycoprotein polypeptide. Preferred antibodyfragments include Fab fragments. In other embodiments the agentcomprises a ligand for the biliary glycoprotein polypeptide, wherein theligand binds only a single biliary glycoprotein polypeptide. Inpreferred embodiments the ligand is fused to an immunoglobulin moleculeor a fragment thereof, or is a soluble biliary glycoprotein molecule orfragment thereof.

According to another aspect of the invention, methods for suppressingspecifically the cytotoxicity or proliferation of killer T cells in asubject are provided. The methods include administering to a subject inneed of such treatment an agent that selectively increases cross-linkingof biliary glycoprotein polypeptides in an amount effective to suppressthe activity of killer T cells in the subject. In certain embodimentsthe agent is an antibody, preferably a monoclonal antibody. In otherembodiments the agent comprises a ligand for the biliary glycoproteinpolypeptide which binds two or more biliary glycoprotein polypeptides.In preferred embodiments, the ligand is fused to an immunoglobulinmolecule or a fragment thereof, or the ligand includes a biliaryglycoprotein polypeptide or fragment thereof.

According to another aspect of the invention, a composition is provided.The composition includes an agent that selectively reduces cross-linkingof biliary glycoprotein polypeptides in an amount effective to enhancecytotoxicity or proliferation of killer T cells in a subject, and apharmaceutically-acceptable carrier. In certain embodiments the agent isan antibody or antibody fragment which binds only a single biliaryglycoprotein molecule. Preferred antibody fragments include Fabfragments. In preferred embodiments, the agent comprises a ligand forthe biliary glycoprotein polypeptide which binds only a single biliaryglycoprotein polypeptide. Preferably such a ligand is fused to animmunoglobulin molecule or a fragment thereof. In certain embodimentsligand is biliary glycoprotein or a fragment thereof. Preferably thecompositions are pharmaceutical compositions.

According to still another aspect of the invention, a composition isprovided which includes an agent that selectively increasescross-linking of biliary glycoprotein polypeptides in an amounteffective to suppress cytotoxicity or proliferation of killer T cells ina subject. The composition also include a pharmaceutically-acceptablecarrier. In some embodiments the agent is an antibody, preferably amonoclonal antibody. In other embodiments the agent includes a ligandfor the biliary glycoprotein polypeptide which binds two or more biliaryglycoprotein polypeptides. Preferably the ligand is fused to animmunoglobulin molecule or a fragment thereof. In other preferredembodiments the ligand is biliary glycoprotein or a fragment thereof.Preferably the compositions are pharmaceutical compositions.

According to still other aspects of the invention, methods for enhancingspecifically the cytotoxicity or proliferation of killer T cells alsoare provided. The methods include contacting a population of killer Tcells with an agent that selectively reduces cross-linking of biliaryglycoprotein polypeptides in an amount effective to enhance thecytotoxicity or proliferation of the killer T cells. The biliaryglycoprotein binding agents are as described above in methods forenhancing killer T cell activity in a subject.

In another aspect of the invention, methods for suppressing specificallycytotoxicity or proliferation of killer T cells are provided. Themethods include contacting a population of killer T cells with an agentthat selectively increases cross-linking of biliary glycoproteinpolypeptides in an amount effective to suppress the cytotoxicity orproliferation of the killer T cells. The biliary glycoprotein bindingagents are as described above in methods for suppressing killer T cellactivity in a subject.

According to another aspect of the invention, an isolated fusion proteinis provided. The isolated fusion protein includes a biliary glycoproteinpolypeptide or a fragment thereof fused to an immunoglobulin molecule ora fragment thereof. The components of the fusion protein can be fuseddirectly, or a linker molecule such as a peptide can be interposedbetween the biliary glycoprotein component and the immunoglobulincomponent. Other polypeptides can be substituted for the immunoglobulincomponent as will be apparent to one of ordinary skill in the art. Incertain embodiments, biliary glycoprotein (or fragment thereof)component of the fusion protein selectively binds a monoclonal antibodyselected from the group consisting of 34B1, 5F4 and 26H7. Preferably thefragment of biliary glycoprotein is selected from the group consistingof the N-domain of CD66a, NA1B1 domains of CD66a, the NA1B1A2 domains ofCD66a. The fragment of the immunoglobulin molecule preferably is the Fcportion of the immunoglobulin molecule.

According to another aspect of the invention, an isolated fusion proteinis provided which includes two or more biliary glycoproteinpolypeptides, or fragments thereof which bind biliary glycoprotein. Thefusion protein is useful for selecting biliary glycoprotein bindingagents which bind two (or more) biliary glycoprotein molecules,particularly those agents which cross-link biliary glycoproteinmolecules.

According to yet another aspect of the invention, methods foridentifying compounds which enhance or suppress killer T cell activityare provided. The methods include contacting a population of killer Tcells which express biliary glycoprotein with a compound that bindsbiliary glycoprotein, and determining the cytotoxicity or proliferationof the population of killer T cells relative to a control. Compoundswhich increase the cytotoxicity or proliferation are compounds whichenhance the killer T cell activity, and compounds which decrease thecytotoxicity or proliferation are compounds which suppress the killer Tcell activity. In certain embodiments, the methods includes the steps ofproviding a biliary glycoprotein polypeptide or a fragment thereof,contacting the biliary glycoprotein polypeptide or a fragment thereofwith a compound, and determining the binding of the compound to thebiliary glycoprotein polypeptide or a fragment thereof. The compound isused in the foregoing methods for testing the increase or decrease ofkiller T cell cytotoxicity or proliferation.

In another aspect of the invention, methods for selectively treating asubject having a condition characterized by aberrant killer T cellactivity are provided. The methods include administering to a subject inneed of such treatment a pharmacological agent which is selective forbiliary glycoprotein, in an amount effective to normalize the aberrantkiller T cell activity.

In the foregoing aspects and embodiments of the invention, preferredkiller T cells include CD4⁺ T cells, CD8⁺ T cells, NK cells, intestinalintraepithelial lymphocytes and peripheral blood T cells. Particularlypreferred killer T cells are CD8⁺ T cells.

The use of the foregoing compositions in the preparation of medicamentalso is provided. In preferred embodiments, the medicament is useful inthe treatment of conditions related to immune system function, includingautoimmune disease, cancer and transplantation.

These and other objects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts identification of three mAbs (34B1, 5F4 and 26H7) whichrecognize IECs but not resting iIELs.

FIG. 2 shows that iIELs and PBTs express an antigen that is recognizedby the 34B1, 5F4 and 26H7 mAbs after activation.

FIG. 3 shows that the N-domain of BGP is the cognate antigen of the34B1-related mAbs.

FIG. 4 shows that the 34B1-related mAbs specifically immunoprecipitateBGP on COS cell transfectants and activated iIELs.

FIG. 5 depicts the specificity of the anti-BGP mAbs for other CD66family members.

FIG. 6 shows an analysis of CD66 isoform expression by activated humaniIELs.

FIG. 7 depicts inhibition of anti-CD3 directed and lymphokine activatedkiller activity of iIELs by anti-BGP antibodies.

FIG. 8 shows inhibition of human allo-mixed lymphocyte reaction byanti-BGP monoclonal antibodies.

DETAILED DESCRIPTION OF THE INVENTION

Evidence for a role of human biliary glycoprotein (BGP, CD66a) as aninhibitory molecule on activated iIELs has been provided through thecharacterization of three monoclonal antibodies raised against anactivated iIEL cell line. These data are especially relevant to iIELs asthey suggest that other molecules such as biliary glycoprotein maycontribute to downregulation of T cell activation in the absence ofCTLA-4. These studies are also relevant to extending the function ofbiliary glycoprotein to an important role in immunoregulation of Tlymphocytes in general given the observation that biliary glycoproteinis also expressed by activated human peripheral blood T cells.

Human biliary glycoprotein is a member of the CEA family ofglycoproteins, part of the immunoglobulin supergene family, and encodedin a large cluster on chromosome 19 (Watt et al., Blood, 84:200-210,1994; Daniel et al., Int. J. Cancer, 55:303-310, 1993; Teixeira et al.,Blood, 84:211-219, 1994; Teixeira, 1996; Barnett et al., Molec. Cell.Biol., 13:1273-1282, 1993). The CEA-cluster is highly related to thegenetically linked, pregnancy specific gene cluster (Thompson et al., J.Clin. Lab Anal., 5:344-366, 1991; Öbrink, Curr Opin Cell Biol.,9:616-626, 1997). The CEA-subgroup of this family is serologicallydefined as CD66a (BGP or C-CAM), CD66b (CGM6), CD66c (NCA), CD66d (CGM1)and CD66e (CEA). These structurally related glycoproteins consist of ahighly homologous membrane distal amino terminal IgV-like N-domain andvariable numbers of membrane distal IgC2-like domains in the case ofBGP, NCA, CGM6 and CEA. In contrast to human CEA, CGM6 and NCA, whichare linked to the membrane by a glycosyl phosphatidyl-inositol anchor,CGM1 and BGP are type 1 transmembrane glycoproteins. Both of the latterexist as isoforms containing short or long cytoplasmic tails.

Biliary glycoprotein, and its mouse and rat homologues C-CAM (Rosenberget al., Cancer Res., 53:4938-4945, 1993; Lin et al., J. Biol. Chem.,264:14408-14414, 1989; Öbrink, BioEssays, 13:227-234, 1991), have beenregarded mainly as molecules which function in cell-cell adhesion thatare expressed primarily by epithelial cells of the gastrointestinaltract and biliary tree, neutrophils and, more recently, B cells. Biliaryglycoprotein also serves as a receptor for mouse hepatitis virus(Williams et al., Proc. Natl. Acad. Sci., 88:5533-5536, 1991) and forOpa proteins of Neisseria species of bacteria (Virji et al., Mol.Microbiol., 5:941-950, 1996). It is of interest that ligation of biliaryglycoprotein on epithelial cells may deliver a negative growth signalwhich may be decreased during tumor formation due to diminishedexpression of biliary glycoprotein (Rosenberg et al., 1993; Kunath etal., Oncogene, 11:2375-2382, 1995; Brümmer et al., Oncogene,11:1649-1655, 1995). Biliary glycoprotein also exhibits a high degree ofalternate transcriptional processing resulting in at least eightpotential alternate transmembrane transcripts. Two of these transcripts,BGPa and BGPb, encode a long cytoplasmic tail of 67 amino acidscontaining two ITIM motifs which suggest a role as inhibitory receptors(Öbrink, 1997). Indeed, this cytoplasmic tail, when tyrosinephosphorylated, is capable of binding SHP-1 in a mouse colon carcinomacell line (Beauchemin et al., Oncogene, 783-790, 1996). Suchinteractions may account for the inhibitory growth effect of thismolecule on epithelial cells.

The studies contained herein describe the unexpected finding that,whereas biliary glycoprotein is constitutively expressed by IECs, it isan activation molecule on T cells adjacent to the epithelium. The studyof peripheral blood T cells, on the other hand, show the unexpectedresult that biliary glycoprotein is constitutively expressed at lowlevels and upregulated by T-cell activation. This difference betweeniIELs and PBTs suggests that biliary glycoprotein expression may beactively suppressed in the epithelium under normal conditions. Theassociation of biliary glycoprotein with the activation state alsoresembles CTLA-4 expression.

Using cytotoxicity, which is a major function of iIELs, as a measure, itappears that biliary glycoprotein on activated iIELs functions as aninhibitory molecule for CD3-directed cytotoxic activity. In this manner,biliary glycoprotein should be considered as a killer inhibitoryreceptor. Although the ligand for biliary glycoprotein on the IEC isunknown, a candidate ligand is biliary glycoprotein itself or anotherCD66 family member in view of the known homophilic and heterophilicinteractions between the CD66 group members (Watt et al., 1994; Oikawaet al., Biochem. Biophy. Res., 186:881-887, 1992; Teixeira et al., 1994;Öbrink, 1997). It therefore can be hypothesized that ligation of biliaryglycoprotein on the IEC by an activated iIEL can serve to function inthe inhibition of IEC growth. Corollarily, the binding of biliaryglycoprotein on the activated iIEL by BGP on the IEC can limit theactivation of the T cell. In tumors of the epithelium where biliaryglycoprotein expression has been observed to be diminished, the growthinhibition effect of the iIEL on the IEC might be lost (Brümmer, 1995).

Thus the invention involves the finding that molecules which bind tobiliary glycoprotein (i.e., “biliary glycoprotein binding agents”) onkiller T cells, such as antibodies, can inhibit or enhance the activityof killer T cells, such as cytotoxicity and/or proliferation. As usedherein, “killer T cells” includes CD4⁺ T cells, CD8⁺ T cells, and NKcells. Biliary glycoprotein binding agents which increase cross-linkingof biliary glycoprotein polypeptides increase the inhibitory signal ofbiliary glycoprotein, thereby suppressing the activity of killer Tcells. Biliary glycoprotein binding agents which decrease crosslinkingof biliary glycoprotein polypeptides decrease the inhibitory signal ofbiliary glycoprotein, thereby enhancing the activity of killer T cells.The invention also embraces molecules which enhance or suppress killer Tcell activity but which do not function according to the cross-linkingproperties described above. For example, a particular biliaryglycoprotein binding agent which suppresses the activity of killer Tcells may bind biliary glycoprotein and increase the inhibitory signalof biliary glycoprotein without increasing cross-linking (e.g., byinducing a conformational change in biliary glycoprotein).

Modulation of killer T cell activity by molecules which bind biliaryglycoprotein expressed on the surface of killer T cells is useful forspecifically enhancing or suppressing an immune response in vivo, whichmay be useful for the treatment of conditions related to immune functionincluding autoimmune disease, cancer, and transplantation (e.g., bonemarrow or organs). Modulation of killer T cell activity also is usefulin in vitro and/or non-therapeutic applications including determiningwhether T cells of a subject are functional (e.g. proliferation and/orcytotoxic functions), to determine if a treatment has rendered killer Tcells non-functional, in experimental models of cancer, autoimmunedisease, and transplantation, e.g., to determine the effects ofincreases or decreases in killer T cell function on particular organs orphysiological processes, and to test for agents which increase ordecrease killer T cell activity. Other uses will be apparent to one ofordinary skill in the art.

The molecules which bind biliary glycoprotein and modulate killer T cellactivity (biliary glycoprotein binding agents) include antibodies andfragments thereof, ligands for biliary glycoprotein, fragments thereofand fusion proteins containing ligands or other biliary glycoproteinbinding molecules. Still other biliary glycoprotein binding agents canbe identified by screening compounds for the ability to enhance orsuppress killer T cell activity, using assays described herein and thoseassays of T cell activity which are standard in the art. Exemplarymethods for preparing fusion proteins useful according to the inventionfor modulating killer T cell activity are described herein; additionalexemplary methods for preparing such fusion proteins are described inU.S. Pat. No. 5,434,131. The molecules which bind biliary glycoproteincan be used alone as a primary therapy or in combination with othertherapeutics as a combination therapy to enhance the therapeuticbenefits of other medical treatments.

The invention also involves agents which bind to biliary glycoproteinand/or fragments of the biliary glycoprotein and induce or suppresskiller T cell activity. In addition to the uses described herein, suchbinding agents can be used in screening assays to detect the presence orabsence of a biliary glycoprotein polypeptide, the presence or locationof iIELs and in purification protocols to isolate iIELs and other killerT cells which express biliary glycoprotein. Likewise, such bindingagents can be used to selectively target drugs, toxins or othermolecules to killer T cells which express biliary glycoprotein. In thismanner, killer T cells which express biliary glycoprotein can be treatedwith cytotoxic compounds, thereby reducing unwanted immune responses.

The biliary glycoprotein binding agents useful according to theinvention, including antibodies and other polypeptides, are isolatedagents. As used herein, with respect to biliary glycoprotein bindingagents, the term “isolated” means that the agents are substantially pureand are essentially free of other substances with which they may befound in nature or in vivo systems to an extent practical andappropriate for their intended use. In particular, the agents aresufficiently pure and are sufficiently free from other biologicalconstituents of their hosts cells so as to be useful in, for example,producing pharmaceutical preparations. Because an isolated biliaryglycoprotein binding agent may be admixed with a pharmaceuticallyacceptable carrier in a pharmaceutical preparation, the biliaryglycoprotein binding agents may comprise only a small percentage byweight of the preparation. A biliary glycoprotein binding agent isnonetheless substantially pure in that it has been substantiallyseparated from the substances with which it may be associated in livingsystems.

According to one embodiment, the biliary glycoprotein binding agent usedin the invention is an intact anti-biliary glycoprotein monoclonalantibody in an isolated form, preferably in a soluble form, or in apharmaceutical preparation. An intact monoclonal antibody, as is wellknown in the art, is an assembly of polypeptide chains linked bydisulfide bridges. Two principle polypeptide chains, referred to as thelight chain and heavy chain, make up all major structural classes(isotypes) of antibody. Both heavy chains and light chains are furtherdivided unto subregions referred to as variable regions and constantregions. As used herein the term “monoclonal antibody” refers to ahomogenous population of immunoglobulins which specifically bind to anepitope (i.e. antigenic determinant) of human biliary glycoprotein.

The invention, therefore, includes the use of antibodies or fragments ofantibodies having the ability to selectively bind to biliaryglycoprotein, particularly as expressed on the cell surface of killer Tcells, such as intestinal intraepithelial lymphocytes and activatedperipheral blood lymphocytes. Antibodies include polyclonal andmonoclonal antibodies, prepared according to conventional methodology.Examples include the monoclonal antibodies 34B1, 5F4 and 26H7 describedin the Examples. Additional antibodies which are reactive with biliaryglycoprotein, particularly those raised against biliary glycoproteinexpressed on killer T cells, can be prepared according to standardmethods.

Antibodies can be prepared by any of a variety of methods, includingadministering protein, fragments of protein, cells expressing theprotein or fragments thereof and the like to an animal to inducepolyclonal antibodies. The production of monoclonal antibodies isaccording to techniques well known in the art. As detailed herein, suchantibodies may be used, for example, to identify tissues expressingprotein or to purify protein. Antibodies also may be coupled to specificlabeling agents for imaging or to cytotoxic agents, including, but notlimited to, methotrexate, radioiodinated compounds, toxins such asricin, other cytostatic or cytolytic drugs, and so forth.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies. Thus, the invention involves polypeptides ofnumerous size and type that bind specifically to biliary glycoprotein.These polypeptides may be derived also from sources other than antibodytechnology. For example, such polypeptide binding agents can be providedby degenerate peptide libraries which can be readily prepared insolution, in immobilized form or as phage display libraries.Combinatorial libraries also can be synthesized of peptides containingone or more amino acids. Libraries further can be synthesized ofpeptoids and non-peptide synthetic moieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention, using methods such as thosedescribed in Hart et al., J. Biol. Chem. 269:12468 (1994). Briefly, oneprepares a phage library (using e.g. m13, fd, or lambda phage),displaying inserts from 4 to about 80 amino acid residues usingconventional procedures. The inserts may represent a completelydegenerate or biased array. One then can select phage-bearing insertswhich bind to biliary glycoprotein or a fragment thereof. This processcan be repeated through several cycles of reselection of phage that bindto biliary glycoprotein or a fragment thereof. Repeated rounds lead toenrichment of phage bearing particular sequences. DNA sequence analysiscan be conducted to identify the sequences of the expressedpolypeptides. The minimal linear portion of the sequence that binds tobiliary glycoprotein or a fragment thereof can be determined. One canrepeat the procedure using a biased library containing insertscontaining part or all of the minimal linear portion plus one or moreadditional degenerate residues upstream or downstream thereof. Thus,biliary glycoprotein can be used to screen peptide libraries, includingphage display libraries, to identify and select peptide biliaryglycoprotein binding agents for modulating killer T cell activity.Preferably, the biliary glycoprotein binding agents are characterized asto their ability to cross-link biliary glycoprotein. Such bindingmolecules can also be used, as described, for screening assays, fordiagnostic assays, for purification protocols or for targeting drugs,toxins and/or labeling agents (e.g. radioisotopes, fluorescentmolecules, etc.) to cells, especially killer T cells, which expressbiliary glycoprotein on the cell surface. Drug molecules that woulddisable or destroy cells which express such biliary glycoprotein or afragment thereof are known to those skilled in the art and arecommercially available. For example, the immunotoxin art providesexamples of toxins which are effective when delivered to a cell by anantibody or fragment thereof. Examples of toxins includeribosome-damaging toxins derived from plants or bacterial such as ricin,abrin, saporin, Pseudomonas endotoxin, diphtheria toxin, A chain toxins,blocked ricin, etc.

Additionally small polypeptides including those containing the biliaryglycoprotein binding fragment (CDR3 region) may easily be synthesized orproduced by recombinant means to produce a biliary glycoprotein bindingagent useful according to the invention. Such methods are well known tothose of ordinary skill in the art. Peptides can be synthesized forexample, using automated peptide synthesizers which are commerciallyavailable. The peptides can be produced by recombinant techniques byincorporating the DNA expressing the peptide into an expression vectorand transforming cells with the expression vector to produce thepeptide.

The sequence of the CDR regions, for use in synthesizing peptide biliaryglycoprotein binding agents, may be determined by methods known in theart. The heavy chain variable region is a peptide which generally rangesfrom 100 to 150 amino acids in length. The light chain variable regionis a peptide which generally ranges from 80 to 130 amino acids inlength. The CDR sequences within the heavy and light chain variableregions which include only approximately 3-25 amino acid sequences mayeasily be sequenced by one of ordinary skill in the art. The peptidesmay even be synthesized by commercial sources.

To determine whether a peptide binds to biliary glycoprotein any knownbinding assay may be employed. For example, the peptide may beimmobilized on a surface and then contacted with labeled biliaryglycoprotein. The amount of biliary glycoprotein which interacts withthe peptide or the amount which does not bind to the peptide may then bequantitated to determine whether the peptide binds to biliaryglycoprotein. A surface having the aforementioned anti-biliaryglycoprotein monoclonal antibodies immobilized thereto may serve as apositive control.

Screening of biliary glycoprotein binding agents also can be carried oututilizing a competition assay. If the biliary glycoprotein binding agentbeing tested competes with an anti-biliary glycoprotein monoclonalantibody, as shown by a decrease in binding of the monoclonal antibody,then it is likely that the agent and the anti-biliary glycoproteinmonoclonal antibody bind to the same, or a closely related, epitope.Still another way to determine whether an agent has the specificity ofthe anti-biliary glycoprotein monoclonal antibodies described above isto pre-incubate the monoclonal antibody with biliary glycoprotein withwhich it is normally reactive (i.e., binds), and then add the agentbeing tested to determine if the agent being tested is inhibited in itsability to bind biliary glycoprotein. If the agent being tested isinhibited then, in all likelihood, it has the same or a functionallyequivalent epitope and specificity as the anti-biliary glycoproteinmonoclonal antibodies.

Using routine procedures known to those of ordinary skill in the art,one can determine whether a biliary glycoprotein binding agent is usefulaccording to the invention by determining whether the agent is one whichmodulates killer T cell proliferation or cytotoxicity in an in vitroassay such as measuring release of TNF from killer T cells or by ⁵¹Crrelease assay (see, e.g., Herin et al., Int. J. Cancer 39:390-396,1987). Other assays are described in the Examples and elsewhere herein.

The polypeptides (e.g. antibodies) and other biliary glycoproteinbinding agents described above can also be used immunotherapeuticallyfor killer T cell sensitive disorders in humans. The term“immunotherapeutically” or “immunotherapy” as used herein in conjunctionwith the biliary glycoprotein binding agents denotes both prophylacticas well as therapeutic administration. Thus, the peptides can beadministered to high-risk subjects in order to lessen the likelihoodand/or severity of a killer T cell sensitive disease, such as a tumor,transplant rejection or autoimmune disease, or administered to subjectsalready evidencing such diseases.

In certain aspects the invention encompasses methods for modulatingspecifically the cytotoxicity or proliferation of killer T cells insitu. The method includes administering to a subject in need of suchtreatment an agent which binds selectively a biliary glycoproteinpolypeptide in an amount effective to enhance or suppress thecytotoxicity or proliferation of the killer T cells in the subject. Asshown in the Examples, the activity of killer T cells is subject tospecific modulation because killer T cells express biliary glycoprotein.Methods for modulating specifically the cytotoxicity or proliferation ofkiller T cells also are provided wherein a population of killer T cellsis contacted with a biliary glycoprotein binding agent. When a biliaryglycoprotein binding agent is administered to a subject or contacted toa population of killer T cells, the inhibitory activity of biliaryglycoprotein is modulated. Biliary glycoprotein binding agents whichincrease or decrease killer T cell activity can be selected using theassays described herein and according to standard killer T cellcytotoxicity and proliferation assays, such as mixed lymphocytereactions, chromium release assays, TNF release assays, and thymidineincorporation assays. It is believed that a monovalent biliaryglycoprotein binding agent will inhibit the inhibitory signal of biliaryglycoprotein by reducing cross-linking of biliary glycoproteinpolypeptides expressed by killer T cells, and that a multivalent biliaryglycoprotein binding agent (having two or more biliary glycoproteinbinding sites) will increase the inhibitory signal of biliaryglycoprotein in killer T cells by increasing cross-linking of biliaryglycoprotein polypeptides expressed by killer T cells.

By definition, the term “in situ” encompasses and includes the terms invivo, ex vivo and in vitro. The compositions of the invention are usefulfor many in vitro purposes. For example, the compositions of theinvention are useful for screening compounds which inhibit killer T cellproliferation or cytotoxicity. Such a screening assay may be performedin vitro by setting up cell proliferation or cytotoxicity assaysincluding a biliary glycoprotein binding agent which increases killer Tcell proliferation or cytotoxicity and a population of killer T cells.Potential killer T cell proliferation or cytotoxicity inhibitors may beadded to the mixture and the effect on proliferation or cytotoxicity maybe measured. Agents which increase proliferation or cytotoxicity ofkiller T cell can be screened using similar assays. Thus methods foridentifying compounds which bind biliary glycoprotein (or modulateactivation of biliary glycoprotein by other molecules such as naturalligands) and enhance or suppress killer T cell activity are providedaccording to the invention. Other in vitro uses, such as researchpurposes, are known to those of ordinary skill in the art.

Ex vivo uses also will be readily identified by those of skill in theart. Ex vivo uses include, for example, the stimulation of proliferationor cytotoxicity of killer T cells which have been removed from amammalian subject and which are subsequently returned to the body of themammalian subject.

The present invention also includes methods for treating a conditioncharacterized by aberrant killer T cell activity, such as cytotoxicityor proliferation. The methods involve the step of administering to asubject having such a condition a pharmacological agent whichselectively binds biliary glycoprotein and modulates a killer T cellactivity, in an amount effective to increase or decrease T-cellproliferation or cytotoxicity.

A “condition characterized by aberrant killer T cell activity” as usedherein is any condition associated with adverse physiologicalconsequences in which an increase or decrease of killer T cell function,embodied by an increase or decrease in killer T cell proliferation orcytotoxicity, results in an improvement of the adverse physiologicalconsequences. Such conditions include disorders of the immune system,such as immunodeficiency, autoimmunity and transplant rejection, as wellas disorders involving undesirable cellular invasion by microorganismsor undesirable cell growth such as tumors.

The biliary glycoprotein binding agents are administered in effectiveamounts. As used herein, an “effective amount” of a biliary glycoproteinbinding agent is an amount which is sufficient to modulate (increase ordecrease) biliary glycoprotein inhibitory function, resulting in amodulation of killer T cell proliferation or cytotoxicity. Modulatingbiliary glycoprotein inhibition of killer T cell activity is sufficientto produce the desired effect in which the symptoms associated with theconditions characterized by aberrant killer T cell activity areameliorated or decreased. Preferably an effective amount of the peptideis a therapeutically effective amount for modulating killer T cellproliferation or cytotoxicity in vivo. Generally, a therapeuticallyeffective amount may vary with the subject's age, condition, weight andgender, as well as the extent of the disease in the subject and can bedetermined by one of skill in the art as a matter of routineexperimentation. The dosage may be adjusted by the individual physicianin the event of any complication. A therapeutically effective amounttypically will vary from about 0.01 mg/kg to about 500 mg/kg, weretypically from about 0.1 mg/kg to about 200 mg/kg, and often from about0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily,for one or several days (depending of course of the mode ofadministration and the factors discussed above).

One of skill in the art can determine what an effective amount of abiliary glycoprotein binding agent is by determining the ability of theagent to modulate killer T cell proliferation or cytotoxicity in an invitro assay. Exemplary assays for measuring the ability of a biliaryglycoprotein binding agent to modulate killer T cell proliferation orcytotoxicity are provided in the Examples and have been discussed above.The exemplary assays are predictive of the ability of a biliaryglycoprotein binding agent to modulate killer T cell activity in vivoand/or ex vivo and, hence, can be used to select agents for therapeuticapplications.

According to the invention, a biliary glycoprotein binding agent may beadministered in a pharmaceutically acceptable composition. In general,pharmaceutically-acceptable carriers for antibodies, antibody fragments,and other biliary glycoprotein binding agents (including small moleculessuch as those derived from combinatorial libraries) are well-known tothose of ordinary skill in the art. As used herein, apharmaceutically-acceptable carrier means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients, i.e., the ability of the agent to modulate killer Tcell activity. Pharmaceutically acceptable carriers include diluents,fillers, salts, buffers, stabilizers, solubilizers and other materialswhich are well-known in the art. Exemplary pharmaceutically acceptablecarriers for peptides are described in U.S. Pat. No. 5,211,657. Theagents of the invention may be formulated into preparations in solid,semi-solid, liquid or gaseous forms such as tablets, capsules, powders,granules, ointments, solutions, depositories, inhalants and injections,for oral, parenteral or surgical administration. The invention alsoembraces pharmaceutical compositions which are formulated for localadministration, such as by implants.

According to the methods of the invention the agents can be administeredby injection, by gradual infusion over time or by any other medicallyacceptable mode. The administration may, for example, be intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous ortransdermal. Preparations for parenteral administration includes sterileaqueous or nonaqueous solutions, suspensions and emulsions. Examples ofnonaqueous solvents are propylene glycol, polyethylene glycol, vegetableoil such as olive oil, an injectable organic esters such as ethyloliate.Aqueous carriers include water, alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobials,antioxidants, chelating agents, and inert gases and the like. Those ofskill in the art can readily determine the various parameters forpreparing these alternative pharmaceutical compositions without resortto undue experimentation.

The methods of the invention also encompass administering biliaryglycoprotein binding agents in conjunction with conventional therapiesfor treating immune system disorders. For example, the methods of theinvention may be practiced simultaneously with conventional treatments.The particular conventional treatment depends, of course, on the natureof the disorder. When, for example, the condition related to aberrantkiller T cell activity is a tumor, a conventional mode of treatment ischemotherapy. The agents of the invention which increase killer T cellactivity (e.g., decrease biliary glycoprotein inhibitory activity) maybe administered in conjunction with chemotherapy in the treatment of thetumor in order to provide enhanced tumoricidal effects. Other immunesystem diseases can be treated concurrently with the biliaryglycoprotein binding agents described herein and other molecules whichbind T cells and affect T cell function, such as CTLA4Ig fusionproteins, as described in U.S. Pat. No. 5,434,131.

The following examples are provided to illustrate specific instances ofthe practice of the present invention and are not to be construed aslimiting the present invention to these examples. As will be apparent toone of ordinary skill in the art, the present invention will findapplication in a variety of compositions and methods.

EXAMPLES

Materials and Methods

Antibodies: The 34B1, 26H7 and 5F4 monoclonal antibodies (mAbs) wereproduced by immunizing BALB/c mice with the activated human mucosallymphocyte line, 191E, as previously described (Russell et al., 1996).Three intraperitoneal injections and a final intravenous injection of5×10⁶ lymphocytes were given at two-week intervals. Three days after theintravenous immunization, splenocytes were isolated and fused with NS1murine myeloma cells in the presence of PEG (m.w. 1450) as describedpreviously. Hybridomas were selected with aminopterin-containing medium,and hybridoma supernatants were screened by indirect immunoperoxidasestaining of frozen intestinal and tonsillar tissue sections. Positivehybridomas were subcloned twice by limiting dilution, and ascitescontaining the antibody was produced by intraperitoneal injection of thehybridoma cells into pristane-treated BALB/c mice. The isotypes of 34B1(IgG1), 26H7 (IgG1) and 5F4 (IgG1) were determined by ELISA using murineisotype-specific mAb (Boehringer Mannheim, Indianapolis, Ind.). W6/32 isa mouse IgG2a mAb specific for human MHC class I (kindly provided by Dr.Jack Strominger, Dana-Farber Cancer Institute, Boston, Mass.). OKT3(IgG2a is a mouse anti-human CD3 mAb (kindly provided by Dr. RobertFinberg, Dana-Farber Cancer Institute, Boston, Mass.). TS2/18 (kindlyprovided by Dr. Llyod Klickstein, Brigham and Women's Hospital) is ananti-CD2 mAb (mouse IgG2a). OKT11 (kindly provided by Dr. EllisReionherz, Dana-Farber Cancer Institute) is an anti-CD2 mAb (mouseIgG29). OKT4 and OKT8 are mouse IgG2a mAbs specific for human CD4 andCD8-α, respectively (obtained from American Type Culture Collection,Bethesda, Md.). UCHT1, directly conjugated to phycoerythrin, is a mouseIgG1 mAb specific for human CD3-ε (Dako, Denmark). The MA22 (CD66a;clone YG-C94G7; IgG1), MA26 (CD66a; clone 4.3.17; IgG1), MA27 (CD66e;clone 26/5/1; IgG2a), MA28 (CD66e; clone 26/3/13; IgG1), MA30 (CD66c;clone 9A6; IgG1), MA41 (CD66b; clone BIRMA17c; IgG1), MA61 (CD66b; clone80H3; IgG1), MA76 (CD66ae; clone 12-140-4; IgG1), MA79 (CD66b; cloneB13.9; IgG1), MA81 (CD66b; clone G10F5; IgG1), MA83 (CD66e; cloneb7.8.5; IgG1), MA84 (CD66de; clone COL-1; IgG2a, MA86 (CD66acde; cloneB6.2; IgG1) and MA91 (CD66e; clone T84.66; IgG1) are mouse mAbs whichwere obtained from the VIth Leukocyte Typing Workshop, Osaka, Japan. Theisotype matched mouse IgG1 negative control mAb was purchased fromKakopatts (Copenhagen, Denmark) or Cappel (West Chester, Pa.). mAbs werepurified by affinity purification and protein-A or G sepharose columnsby standard methods.

Cells and Cell Lines: Peripheral blood mononuclear cells (PBMC) wereobtained by Ficoll-Hypaque gradient centrifugation using standardmethods. Peripheral blood T cells were stimulated by cultivating PBMCsfor 72 hours at 37° C. in RPMI-1640 (Gibco, Grand Island, N.Y.)containing penicillin/streptomycin (100 units/ml), 10 mM Hepes pH7.4,10% fetal calf serum and 1 μg/ml phytohemagglutin-P (PHA-P) (MurexDiagnostics, Dartford, England). Human iIEL cell lines EEI-10 (smallintestine), EEI-5 (small intestine) and CLI (large intestine) weregenerated as previously described (Christ et al., Immunol. Let.,58:159-165, 1997) and maintained by stimulation with 1 μg/ml PHA-P cellsin RPMI-1640 containing 10% human serum (type AB, Sigma, St. Louis,Mo.), 5 units/ml rIL-4 (Genzyme, Cambridge, Mass.) and 2 nM rIL-2 (akind gift from Ajinomoto Co., Ltd., Japan) and irradiated PBMC as feedercells. HT29 is a human intestinal epithelial cell (IEC) line obtainedfrom the ATCC. COS is a monkey kidney fibroblast cell line. These lattercell lines were maintained in RPMI-1640 containing 10% heat-inactivatedfetal calf serum (Gibco), penicillin and streptomycin, nonessentialamino acids and 10 mM HEPES (complete medium) at 37° C. in 5% CO₂.

Transfectants. The BGPx′ molecule was constructed as follows. The Nterminal domain and the transmembrane/cytoplasmic domains of human BGPcwere each amplified separately by PCR with the primer pairs BGPAMP-S:caucaucaucauaagcttatggggcacctc (SEQ ID NO:1) and NTM-AS:gccattttcttggggcabctccgggtatac (SEQ ID NO:2); NTM-S:gtatacccggagctgccccaagaaaatggc (SEQ ID NO:3) and BGPTRANS-CYT-AS:cuacuacuacuaagactatgaagttggttg (SEQ ID NO:4), respectively, where theNTM primers were hybrids of the 3′ end of the N terminal domain and the5′ end of the transmembrane domain. The PCR reaction consisted of 5 μl10×Taq buffer (10 mM Tris-HCl pH8.3, 50 mM KCl, 0.1% gelatin), 3 μl 1.5mM MgCl₂, 1 μl 200 μM of each dNTP, 1 μM of each primer, 1 Unit Taqpolymerase and 1 μg cDNA in a final volume of 50 μl. The PCR reactionwas carried out at conditions of 94° C. for 10 min, followed by 25cycles of 94° C. for 1 min, 55° C. for 1 min, and 72° C. for 2 min, plusa final extension time of 10 min at 72° C. After passing the PCRproducts through S-300 columns, 5 μl of each PCR product were used in asecond PCR. After the PCR products had annealed, the BGPAMP andTRANS-CYT AS primers were added to the reaction mix and the PCR reactioncarried out as described above. The resulting PCR product was clonedinto the pAMP 1 vector using the CloneAMP system as detailed by themanufacturer (Gibco-BRL, Gaithersburg, Md.), transformed into DH5αcompetent bacteria and positive transformants selected by PCR. Theresulting BGPx′ cDNA was extracted and sequenced by standard methods.The BGPx′ cDNA was digested with EcoRI and NotI restriction enzymes andsubcloned into the pcDNA1/Amp vector (Invitrogen, Carlsbad, Calif.). TheBGPx′ cDNA in this vector and the pSV2neo plasmid (Clontech, Palo Alto,Calif.) were linearized with XhoI and BamHI, respectively, andelectroporated into CHO cells at a ratio of 15:1, selected in G418 andon the FACS cell sorter to create a stable CHO-BGPx′ cell line asdescribed earlier (Watt et al., 1994). CHO cells stably transfected withBGPx′, Neomycin (Neo), BGPc (Watt et al., 1994)) and BGPa (Oikawa etal., Biochem. Biophys. Res. Commun., 186:881-887, 1992) and HeLa cellsstably transfected with CEA, CGM1, NCA and CGM6 have been previouslydescribed (Daniel et al., 1993).

Flow Cytometry: For one color immunofluorescence, approximately 1×10⁶cells were stained with 1 μg of the primary mAb for 30 minutes on ice.Cells were then washed with phosphate buffered saline (PBS) containing2% fetal calf serum or 0.2% bovine serum albumin and 0.02% sodium azide(WB) followed by incubation for 30 minutes with 1 μg of agoat-anti-mouse fluorescein isothiocyanate labeled secondary antibody(Zymed, San Francisco, Calif.) diluted in WB. The cells were washed withWB and either examined fresh or resuspended in 1% paraformaldehyde inPBS. The stained and fixed cells were analyzed on either an Epics V flowcytometer (Coulter, Hialeah, Fla.) or a FACSCalibur (Becton-Dickinson,Sunnyvale, Calif.). For two color immunofluorescence, staining wasperformed as described above except that after incubation with primaryconjugate, the stained cells were blocked for 20 minutes in PBScontaining 20% normal mouse serum followed by incubation with a directlyconjugated mAb.

Immunohistochemistry: Tissue samples were mounted in OCT compound (AmesCo., Elkart, Ind.), frozen in liquid nitrogen or in a cryostat andstored at −70° C. Frozen tissue sections 4 μm thick were fixed inacetone for 5 minutes, air dried, and stained by an indirectimmunoperoxidase method (Canchis et al., Immunology, 80:561-569, 1994)using avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame,Calif.) and 3-amino-9-ethylcarbazole (Aldrich Chemical Co., Inc.,Milwaukee, Wis.) as the chromogen.

COS cell expression cloning: A cDNA library was constructed in the pCDM8vector using poly(A)⁺ RNA from resting and activated human PBT and fromNK cells in the vector pAEXF (Hall et al., Proc. Natl. Acad. Sci.,93:11780-11785, 1996). For the first round of selection, COS cells weretransfected via the DEAE-Dextran procedure (Seed et al., Proc. Natl.Acad. Sci., 84:3365-3369, 1987) with 0.2 μg of library DNA per 100 mmdish. After 40 hr, cells were harvested, incubated with 34B1 mAb (1:500dilution of ascites), washed, and panned on anti-IgG1 coated plates aspreviously described (Seed et al., 1987; Freeman et al., J. Immunol.,143:2714-2722, 1989). Episomal DNA was prepared from adherent cells,re-introduced into E. coli, transfected into COS cells by polyethyleneglycomediated fusion of spheroplasts (Seed et al., 1987), and thepanning with 34B1 mAb repeated. Individual plasmid DNAs were transfectedinto COS cells via the DEAE-Dextran procedure and analyzed after 72 hrfor cell surface expression by indirect immunofluorescence and flowcytometry.

Radiolabeling, immunoprecipitation and electrophoresis: COS cells, 96hours after transient transfection, were removed noenzymatically fromplastic Petri dishes and labeled with Na-[¹²⁵I] by the lactoperoxidasecatalyzed method as previously described (Balk et al., Science,265:259-262, 1994). After washing, radiolabeled cells were lysed inimmunoprecipitation buffer (IPB) containing 150 mM sodium chloride, 50mM Tris pH 7.8, 10 mM Iodoacetamide, 1 mM EDTA (1 mM PMSF and 1 μg/mleach of leupeptin, pepstatin, aprotinin, and chymostatin with 1% NonidetP40 as a detergent). Cells were lysed on ice for 30-60 minutes followedby centrifugation at 14,000 g for 15 minutes at 4° C. The supernatantfrom the centrifugation was then centrifuged at 100,000 g at 4° C. in aTL-100 ultracentrifuge. The supernatant from this centrifugation wasincubated with 20 μl of packed protein-A Sepharose beads and rockedovernight at 4° C. After preclearing with either an irrelevant isotypematched mAb or normal mouse serum coupled to either protein-A orprotein-G Sepharose beads, specific immunoprecipitations were performedby rocking overnight at 4° C. with mAbs coupled to protein-A or Gsepharose (Pharmacia, Piscataway, N.J.) and the beads washed with IPBcontaining detergent. For N-glycanase digestions, immunoprecipitateswere suspended in 0.25 M Na—HP0 ₄ and 1 mM EDTA after boiling in 0.8%β-mercaptoethanol and 0.5% SDS. Immunprecipitates were then eithertreated with 1 unit of N-glycanase (Genzyme, Boston, Mass.) or mocktreated at 37° C. overnight and resuspended in Laemmli buffer containingreducing agents. Solubilized immunoprecipitates were then resolved in12.5% polyacrylamide gels in the presence of SDS.

Production of soluble recombinant proteins: Details of the pIG plusvector (R&D Systems Europe Ltd., Abingdon, UK) containing the Fc genomicfragment of human IgG1 and incorporating the hinge (H), CH2 and CH3domains of the construction of the CD66a-Fc soluble proteins containingthe N, NA1B1 and NA1B1A2 extracellular domains, Muc-18-Fc and NCAM-Fchave been described previously (Teixeira et al., 1994; Buckley et al.,J. Cell. Sci., 109:437, 1996; Teixeira, 1996). NCAM-Fc, Muc-18-Fc (R&DSystems) and the CD66-Fc (N-Fc, NA1B1-Fc and NA1B1A2-Fc) cDNAs weretransfected into COS cells and the secreted soluble protein purified onprotein-A Sepharose as previously described (Watt et al., 1994; Teixeiraet al., 1994).

Analysis of antibody binding to soluble recombinant proteins: Aspreviously described (Teixeira et al., 1994), ninety-six well flatbottom microtiter plates (Immulon 3) were coated with 100 μl anti-humanFc (Sigma, St. Louis, Mo.) at a final concentration of 1 μg/ml in 10 mMTris-HCl pH 8 overnight at 4° C. Wells were washed four times, blockedfor 1 hr at room temperature with 0.25% BSA, 0.05% Tween 20 in PBS (pH7.4), and coated overnight at 4° C. with 50 μl of soluble Fc constructat a final concentration of 10 μg/ml in PBS. After washing the wellsfour times with PBS, 50 μl of mAb at varying dilutions in PBS were addedper well for 1-2 h at room temperature. Wells were washed with PBS fourtimes and 50 μl of 1:4000 dilution of alkaline phosphatase conjugatedgoat anti-mouse Ig (Boehringer-Mannheim) in PBS were added per well for1 h at room temperature. The wells were washed four times with PBS and200 μl paranitrophenyl phosphate substrate were added (Sigma) anddeveloped for 15-45 min at room temperature. The absorbance at 405 nmwas determined. All experiments were carried out in triplicate andrepeated at least twice.

Redirected Lysis: Cytotoxicity was evaluated as previously described(Probert et al., J. Immunol., 158:1941-1948, 1997). Briefly, the P815mouse mastocytoma cell line was labeled with 100 μCi [⁵¹Cr] (New EnglandNuclear, Boston, Mass.) at 37° C. for 30 minutes. 2×10³ radiolabeledcells, in 100 μl complete medium, were added to 100 μl of varyingconcentrations of effector T cells in 100 μl of complete medium intriplicate in a 96 well V-bottom plate. Prior to addition of targetcells, the effector cells were incubated for 20 minutes at roomtemperature with the OKT3 mAb (200 ng/ml of purified antibody) and/or34B1, 26H7 or 5F4 mAbs (either 1:600 dilution of ascites or varyingconcentrations of purified antibody). After 5 hours, 100 μl ofsupernatant were removed for analysis in a γ-counter (LKB Wallac CliniGamma 1272, Finland). Spontaneous and maximal release were measured byincubating target cells with medium or 1% Nonidet-P40, respectively.Percent cytotoxicity was calculated using the formula (experimentalrelease−spontaneous release)×100/(maximal release−spontaneous release).

Statistics: Differences between samples were evaluated with anon-paired, Student's T-test using the Sigma Stat (Jandel Scientific,San Rafael, Calif.) program.

Example 1 Constitutive Expression of the 34B1-Related Antigen on theCell Surface of Normal Human IECs

During the development of iIEL specific mAbs, which were obtained byimmunizing mice with an iIEL T-cell line from human small intestinepropagated in vitro, it was noted that a certain fraction of the mAbsstained IECs as shown by immunohistochemistry of normal human smallintestine. Three of these mAbs and the characterization of the antigenthat they recognized were of particular interest, as described below.

FIG. 1 depicts identification of three mAbs (34B1, 5F4 and 26H7) whichrecognize IECs but not resting iIELs. Panels A-C show immunohistology ofnormal human large intestine stained with the 34B1 (panel A), 5F4 (panelB) and 26H7 (panel C) mAbs with binding detected by subsequentincubation with a goat anti-mouse horseradish peroxidase conjugatedantibody as described in the Materials and Methods. The precipitatedbrown reaction product indicates specific staining on the enterocyte.[Magnification: 20×]. Staining with normal mouse serum was negative(data not shown).

Staining of human intestinal tissue sections showed that these threemAbs (34B1, 26H7 and 5F4) only stained IECs (FIG. 1). This in vivotissue staining with these antibodies appeared to be on the cell surfaceas confirmed by flow cytometry analysis of a normal human IEC line,HT29. Since these three antibodies did not stain iIELs in situ, asdetermined by immunohistochemistry (FIG. 1), or immediately afterisolation as determined by flow cytometry (data not shown), it wassuspected that iIELs, activated during the process of in vitrocultivation, expressed neoantigens that were constitutively expressed byIECs.

Example 2 The 34B1-Related Antigen is an Activation Antigen on NormalHuman iIELs and Peripheral Blood T Cells

To determine whether iIEL cell lines expressed neoantigens that wereshared with IECs, the staining of iIELs was examined after in vitrocultivation with PHA-P. One color flow cytometry analysis was performedof an activated iIEL cell line derived from the small intestine, EEI-5,as described in FIG. 1. As shown in FIG. 1A, iIELs in situ and freshlyisolated iIELs (data not shown) did not stain with the 34B1, 26H7 and5F4 mAbs. However, after maintenance in vitro as continuous cell lineswith PHA-P activation every 10-14 days, all of the iIELs expressed theantigen recognized by these three mAbs (FIG. 2A). Staining of an iIELT-cell line established from the small intestine, EEI-5, which was 90%CD8⁺ and 10% CD4⁺ as shown in FIG. 1B indicates that all iIEL expressedthe antigen recognized by the three mAbs after in vitro activation. Thethree mAbs exhibited slightly different staining patterns suggestingthat they either recognized a different molecule or a different epitopeon the same molecule. Similar observations were made with an iIEL T cellline prepared from the large intestine, CLI, which was 40% CD8⁺, 30%CD4⁺ and 30% double negative (CD4⁻CD8⁻) consistent with the in vivophenotype of iIELs in this tissue site (Lundqvist et al., Int. Immunol.7:1473-1480, 1996; data not shown).

These characteristics were, however, not confined to iIELs since PBTsexpressed the 34B1-related antigen and upregulated this expression afterstimulation with PHA-P in vitro. FIG. 2B shows the two-colorfluorescence analysis of normal PBTs, before and after three days ofstimulation with 1 μg/ml PHA-P, with the 34B1, 5F4 and 26H7 mAbs aftergating on the CD3-positive lymphocytes and after subtracting backgroundstaining with directly conjugated, isotype matched control antibody. Thethick black line shows staining of PBTs without PHA-P stimulation andthe thin black line (arrow) with PHA-P stimulation. Prior to PHA-Pstimulation, a discrete population of T cells exhibited increasedstaining with the 34B1, 5F4 and 26H7 mAbs (FIG. 2B). After PHA-Pstimulation, a small but significant shift in the intensity of stainingof the entire population with all three mAbs was observed. Thus, the34B1-related antigen is an activation antigen on both normal human Tcells in the intestinal epithelium and peripheral blood.

Example 3 The 34B1-Related Antigen is Expressed on Epithelial Cells, BCells and Granulocytes in a Wide Variety of Organs

The data above suggested that the 34B1-related antigen was expressed byepithelial cells of the intestine and activated T cells. A limited organsurvey of the distribution of this cellular expression was thereforeexamined. As can be seen in Table 1, the antigen(s) recognized by the34B1, 26H7 and 5F4 mAbs were similarly expressed within a wide varietyof tissues suggesting that the molecule(s) of interest had a functionalrole in diverse organ systems. All three mAbs consistently recognized anantigen on epithelial cells of the small and large intestine, biliarytree, kidney, skin and thymus. In addition, scattered granulocytes inseveral organs and cells within germinal centers of tonsils, which wereconsistent with B cells, also stained positive. The staining ofgranulocytes was confirmed by immunohistochemical analysis of peripheralblood granulocytes (data not shown). Thus, these results, together withthe phenotypic studies described above, suggested that the 34B1-relatedantigen(s) was primarily expressed by a wide variety of epithelial celltypes, B and T lymphocytes, granulocytes and natural killer (NK) cells,based upon staining of an NK cell line (data not shown).

TABLE I Tissue Staining of the 34B1-Related mAbs Tissue Staining PatternKidney Proximal tubules (+) Glomeruli (+) Endothelium (+) Liver Biliarycanaliculi (+) Bile ducts (+): luminal surfaces Lymph Node Sinusoids(+): Granulocytes/Platelets Skin Epidermis (−) Eccrine/Sweat glands (+)Small Intestine Enterocyte (+): Villous > Crypt Goblet cells (−)Granulocytes (+) Thymus Hassal corpuscles (+) Tonsil Germinal centers(+) Epithelium (+)Staining of all tissues is shown as described in the Materials andMethods. For each tissue, cellular staining was graded as either absent(−) or present (+) as defined by a pathologist.

Example 4 Identification of the 34B1-Related Antigen as BiliaryGlycoprotein (BGP)

To identify the molecule recognized by the 34B1-related mAbs, the 34B1mAb was used to clone the cDNA which coded for the cognate antigen ofthe 34B1 mAb by COS cell expression cloning after transfection with amixture of three cDNA libraries from resting and activated human PBTsand NK cells. These cDNA libraries were utilized because previousstudies showed expression of the antigen recognized by the 34B1, 26H7and 5F4 mAbs in these cell types. Transiently transfected COS cells weresubjected to three rounds of immunoselection and panning with the 34B1mAb. After the third round of panning, 17 of 50 random-selected E. colitransformants contained plasmids with a 3.3 kb-insert. The inserts inthese plasmids were similar by restriction digest analysis. COS cellstransfected with these plasmids were specifically stained with the 34B1mAb. One of these plasmids, pPAN3.1, was selected for furthercharacterization.

This plasmid directed the translation, when transfected into COS cells,of a 120-kD glycoprotein which was specifically recognized by the 34B1and 5F4 mAbs and that resolved as a major band of approximately 70-kDand several minor bands of lower molecular weight after digestion withN-glycanase (FIG. 4). Cell surface proteins of COS cells transientlytransfected with the pPAN3.1 vector encoding BGPb (lanes a-c) or thepCDM8 vector (lanes d and e) and the activated iIEL cell line, EEI-10(lanes f and g) were radiolabeled with [¹²⁵I] and immunoprecipitatedwith either the 34B1 mAb (lanes b, c, d, e, f and g) or normal mouseserum (lane a) and the immunoprecipitates resolved under reducingconditions with (lanes c, e and g) or without (lanes a, b, d and f)prior N-glycanase treatment. Identical observations were made with the5F4 and 26H7 mAbs (data not shown). A similar glycoprotein wasimmunoprecipitated from radiolabeled cells surface iIEL proteins by allthree mAbs (FIG. 4). Complete DNA sequencing of this cDNA on bothstrands revealed a sequence that was 97% identical to the ‘b’ splicevariant of BGP or CD66a (Gen Bank accession #X14831) with all thedifferences occurring outside the coding region. Since the cDNApredicted a polypeptide backbone of 58 kD, the data in FIG. 4 suggestthat several of the carbohydrate modifications were relatively resistantto N-glycanase digestion.

BGPs are members of the immunoglobulin supergene family that consists ofan N-terminal immunoglobulin V-(IgV) related domain, that is highlyhomologous to the N-domains of other carcinoembryonic antigen (CEA) orCD66 family members, followed by several IgC2-related domains A1 and B1,and the A2, Y or Z domains which are unique to BGP isoforms (Watt etal., 1994; Oikawa et al., 1992; Teixeira, 1994; Barnett et al., 1993;Thompson, 1991).

In order to confirm that the 34B1-related mAbs were reactive with BGPand to define the specific protein domain to which these mAbs weredirected, the antibodies were tested in a binding assay with Fc-fusionproteins containing either the N-domain of CD66a, NA1B1 domains ofCD66a, the NA1B1A2 domains of CD66a, and N-CAM (CD56) as a negativecontrol. FIG. 3A is a schematic diagram of the Fc-fusion proteins usedin the ELISA to test the mAbs as described in the Materials and Methods.Fc-fusion proteins containing the N, NA1B1 and NA1B1A2 domains of CD66aor N-CAM (CD56) as a negative control were tested in an ELISA asdescribed in the Materials and Methods with the 34B1, 5F4 and 26H7 mAbsin comparison to the positive control antibodies, MA22, MA76 and MA26(FIG. 3B). As can be seen in FIG. 3, these studies confirmed therecognition of BGP (CD66a) by the three mAbs and showed that all threemAbs reacted with the N-domain.

In order to further confirm that the cognate antigen of the 34B1-relatedmAbs was BGP, the three mAbs were tested for their ability to stain CHOcells stably transfected with several splice variants of BGP (BGPa, BGPcand BGPx′) and HeLa cells transfected with other members of the CD66serologic cluster including CD66b (CEA gene related member 1, CGM1),CD66c (CEA gene related member 6, CGM6), CD66d (Nonspecific crossreacting antigen, NCA) and CD66e (CEA). FIG. 5 shows the flow cytometricanalysis of BGPa, BGPc and BGPx′ transfectants of CHO, and CEA, NCA,CCGM6 and CGM1 transfectants of HeLa cells in comparison to the mock(Neo) transfectants after staining with either the 34B1, 5F4 and 26H7mAbs or isotype matched IgG1 antibody as a negative control. Alltransfectants were positively stained with control mAbs specific for thetransfected cDNA (data not shown). Except for CGM1 (CD66b), the 34B1 mAbstained all the CD66 family members tested including all of the CD66asplice variants. The 26H7 and 5F4 mAbs, however, only stained the CD66asplice variants suggesting they were likely specific for the N-domain ofthis molecule. These results, together with those from phenotyping andthe distribution of BGP on epithelial cells, granulocytes, B cells, Tcells and NK cells described above and previously reported (Thompson etal., 1991; Möller et al., 1996), clearly identify the N-domain of BGP asthe cognate antigen of the 34B1, 26H7 and 5F4 mAbs and that, inaddition, the 34B1 mAb recognizes other CD66 forms.

Example 5 iIELs Express Only CD66a Isoform

Activated human iIELs were stained with a panel of mAbs that have beenpresented at the Sixth International Workshop on Monoclonal Antibodiesand which are specific for all of the CD66 isoforms. The human iIEL cellline from small intestine, EEI-10, was stained 8 days after activationwith a series of anti-CD66 mAbs as described in the Materials andMethods. Each panel shows an overlay of the CD66 specific mAbs with thestaining obtained with normal mouse serum as a negative control. Thespecificity of the mAbs for the CD66 isoforms is indicated in thepanels. Activated iIELs do not express any CD66 isoforms other thanCD66a based upon staining with a large panel of mAbs specific forCD66a-e (mAbs MA27, MA28, MA30, MA41, MA61, MA76, 79, MA81, MA83, MA84,MA86 and MA91 (FIG. 6).

To draw a further similarity between BGP and ITIM containing KIRs, theeffects of the CD66a specific mAbs on the pattern of tyrosinephophsorylated proteins after ligation of CD3 was assessed with theEei-10 cell line. iIELs (1×10⁶) were incubated in 100 μl RPMI-1640containing 0.1% bovine serum albumin (Sigma) with 100 ng/ml OKT3 mAb andeither 10 μg/ml of the 5F4 mAb or 10 μg/ml normal IgG1 in the presenceof 20 μg/ml goat anti-mouse IgG antibody (Pierce, Rockford, Ill.) ascrosslinker. After either 2 or 8 minutes incubation at 37° C., thereaction was stopped with 1 ml PBS containing 10 mM Na₂VO₃ (Sigma), thepellet incubated on ice for 30 minutes in 100 μl lysis buffer containing0.2×PBS, 100 μM Na₂VO₃, 1 mM PMSF, 5 mM iodoacetamide and 20 μg/mlaprotinin and Laemmli buffer added with reducing agents. The lysateswere boiled for 5 minutes, resolved by SDS-PAGE on a 10% gel, Westerntransferred to Immobilon filters, immunoblotted with the PY20 mAb(Zymed, San Francisco, Calif.) and developed with a horseradishperoxidase conjugated goat anti-mouse antibody (Zymed) and enhancedchemiluminescence. Cross-linking of CD3 with the OKT3 mAb in thepresence of the 5F4 mAb, in comparison to an isotype controlledantibody, resulted in a significant alteration in the kinetics and levelof phosphorylation suggesting that a phosphatase was activated as aconsequence of BGP cross-linking.

Example 6 BGP Functions as a Killer Inhibitory Receptor for iIELs

The observation that BGP was expressed on activated iIELs as defined bystaining with the BGP-specific mAbs, 34B1, 26H7 and 5F4 was unique andunexpected since BGP has previously been primarily viewed as a moleculeexpressed on epithelial cells and granulocytes and involved in cell-celladhesion and regulation of epithelial cell growth. The function of BGPon iIELs and T cells in general was, however, unknown. Importantly, thecytoplasmic tail of the BGPa and BGPb splice variants, but not CD66b-e,contain two ITIM domains within the cytoplasmic tail separated by 21amino acids raising the possibility that BGP might function as aninhibitory molecule on T cells (Beauchemin et al., 1996; Öbrink, 1997).In order to define the role of the BGP antigen in T cell function, theeffects of the three mAbs on the function of iIELs was examined.

A major function of activated iIELs is as cytolytic effector cells.Therefore, the effects of the three mAbs on the cytolytic function ofiIELs was examined in a redirected lysis assay, the results of which aredepicted in FIG. 7. The anti-CD3 directed lysis (OKT3) of the KJ-3 iIELcell line was examined as described in the Materials and Methods in theabsence or presence of either the CD66a specific mAbs, 5F4 or 26H7, oran isotype matched IgG1 antibody at a concentration of 100.0 μg/ml ateffector:target ratios of 100:1, 50:1 and 25:1. The cytolysis in theabsence of added antibodies (medium) and presence of the test antibodiesalone (26H7, 5F4 and control IgG1) are also shown. The standard error ofthe mean for each measurement is indicated. The data presented in FIG. 7is representative of six experiments. When an iIEL cell line, EEI-10,derived from normal human small intestine, was examined in a redirectedlysis assay with the P815 mouse mastocytoma cell line as a target cell,significant cytotoxicity was elicited with, but not without, anti-CD3cross-linking using the OKT3 mAb (FIG. 7). Neither the 34B1, 26H7 nor5F4 mAbs at a variety of different concentrations were able to stimulatecytolysis of the P815 cell line indicating that cross-linking BGP didnot directly activate iIELs. However, cross-linking BGP with all threeanti-BGP mAbs resulted in a significant inhibition of the anti-CD3directed cytolysis of the P815 cell line in comparison to an isotypematched IgG1 control antibody which exhibited no inhibition. The 5F4 mAbinhibited the lysis by 22%, 35% and 38% at effector:target ratios of100:1, 50:1 and 25:1, respectively. The inhibition by the controlantibody at similar effector:target ratios was −4%, 5% and 5%. Moreover,the anti-CD3 directed cytolysis was not inhibited by a CD2-specific mAb,TS 2/18, which would be expected to inhibit CD58-like interactions withthe P815 cell line, suggesting that the inhibition by the anti-CD66amAbs was not likely due simply to an effect on adhesion. Thus,cross-linking of BGP inhibited the anti-CD3 directed cytolytic activityof iIELs.

When iIELs were harvested early after cytokine treatment, a significantamount of cytolysis was observed against the P815 cell line consistentwith cytokine induced killer activity (lymphokine activated killeractivity, LAK), a property previously described for iIEL. To testwhether this cytolytic activity was also subject to inhibition by theanti-BGP mAbs, the iIEL cell line was exposed to the P815 cell line astarget in the presence or absence of the BGP specific mAbs, a pool ofthe BGP specific mAbs or normal IgG1. The effects of the anti-BGPspecific mAbs 34B1, 26H7 and 5F4 on cytolysis of the p815 cell line bythe EEI-10 iIEL cell line was examined at an effector target ratio of25:1. The cytolysis of the p815 cell line was not affected by theirrelevant IgG1 antibody at either 2 μg/ml or 6 μg/ml. The inhibition ofthe cytotoxicity was significant for all anti-BGP antibodies (5F4,p=0.15; 26H7, p=0.048, 34B1, p=0.004; pool, p=0.0016). The anti-BGP mAbsinhibited the cytolytic activity of the iIEL cell line by as much as 70%using the pool of mAbs at 2 μg/ml each, and up to 50% inhibition atdoses of individual mAbs of 2 μg/ml, indicating the lymphokine activatedkiller activity of iIELs was also subject to inhibition by the anti-BGPantibodies.

Example 7 Anti-BGP Antibodies can Inhibit the BGP Inhibitory Signal inan Allogenic Mixed Lymphocyte Reaction

Human peripheral blood mononuclear cells (donor A) were irradiated (5000Rads) and cococultivated with an equivalent number of nonirradiatedperipheral blood mononuclear cells prepared by Ficoll-Hypaque gradientcentrifugation at a total concentration of 2×10⁵ cells per well of a96-well U-bottom plate in a total volume of 200 μl in quadruplicate.After 96 hours, 0.5 μCi of [³H]-thymidine was added per well for 18hours of incubation and the plates harvested and counted. The cultureconditions contained no additives (medium) or various concentration ofdiluted ascites (mouse anti-human BGP monoclonal antibodies 34B1 and5F4) or normal mouse serum. The experimental data depicted in Table IIare representative of 5 experiments. The mean ±SE is presented. Theaugmentation of T cell proliferation in the presence of anti-BGPantibodies is consistent with the inhibition of a BGP inhibitory signal.

TABLE II Allogeneic Mixed Lymphocyte Reaction Treatment CPM Medium16,330 ± 2,131 mouse anti-human BGP (34B1) 1:125 41,246 ± 1,779 1:25054,578 ± 2,437 1:500 34,987 ± 1,545 1:750 33,978 ± 1,036 1:1000 31,522 ±1,087 mouse anti-human BGP (5F4) 1:125 35,330 ± 1,615 1:250 33,191 ± 8831:500 27,862 ± 2,635 1:750 23,511 ± 2,365 1:1000 38,826 ± 3,103 Normalmouse serum (negative control) 1:125 18,491 ± 544 1:500 17,957 ± 7721:1000 19,343 ± 471

FIG. 8 shows the results of similar experiments. Stimulator (irradiatedperipheral blood mononuclear cells) and responder (nonirradiatedperipheral blood mononuclear cells) from two unrelated human studysubjects were cocultivated in at 2×10⁵ cells per well each in a 96-wellflat bottomed plate in the presence or absence of eitherphytohemagglutinin-P (PHA; 1 μg/ml), irrelevant IgG1 at variousconcentrations (1-100 μg/ml) or the anti-CD66a specific monoclonalantibodies 34B1, 26H7 or 5F4 at various concentrations (1-100 μg/ml).After four days, 1 μCurie of ³H-thymidine was added per well for thelast 18 hours of incubation and the plates harvested for assessment ofproliferation. The y-axis shows the counts per minute. The S.E.M. isshown for each measurement.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

All of the references described herein are incorporated by reference.

1. A method for the treatment of an autoimmune disease or transplantrejection comprising administering to a subject in need of suchtreatment an antibody or an antigen-binding fragment thereof, whereinsaid antibody or antigen-binding fragment thereof binds two or morebiliary glycoprotein polypeptides.
 2. The method of claim 1, wherein theantigen-binding fragment is a F(ab)₂ fragment.
 3. The method of claim 1,wherein the antibody is a monoclonal antibody or an antigen-bindingfragment thereof.